Tumor cell evasion from regulated cell death is a hallmark of cancer [1]. Davern et al. now identify resistance to mitochondrial apoptosis to represent the common mechanism by which persister cells that survived either immunotherapy- or irradiation-treatment rely on to proliferate. Therefore, they discovered the persister cells´ Achilles heel for future treatments with BH3-mimetics [2].

Acute tubular necrosis (ATN) is a central driver of nephron loss and progression to kidney failure. Dissecting the cellular pathways that govern tubular necrosis requires reliable systems to model and quantify cell death. This chapter introduces methodologies for isolating murine renal tubules ex vivo and describes a panel of assays-including biochemical, molecular, and imaging approaches-that enable the detection of regulated necrosis in kidney tubules. We provide theoretical background, practical workflows, examples of applications.

Necrotic zones in tissues occur in a wide variety of diseases. Ferroptosis, an iron-promoted necrosis driven by lipid peroxidation, has been identified as a key cell death modality in these conditions. Cells undergoing ferroptosis are unique in that they can induce death in their neighbours. Here we review salient aspects of ferroptosis propagation on the molecular, cellular and tissue levels. Cell death propagation is restricted by several ferroptosis-suppression systems. Glutathione peroxidase 4 (GPX4) and ferroptosis-suppressor protein 1 (FSP1) are now well established as master regulators, but various additional systems dictate ferroptosis sensitivity. We discuss how these mechanisms contribute to the suppression of cell death propagation, and how they cause various tissues to be more or less resistant to ferroptosis propagation. Understanding tissue-specific dynamics is critical to interpret the beneficial effects and limitations of future therapeutic approaches for ferroptosis-driven diseases.

Heme carries oxygen and is critical for the control of redox reactions. In this issue of Cell, Lewis and Gruber et al. demonstrate how low concentrations of heme destabilize complex IV of the respiratory chain to release copper and kill acute myeloid leukemia cells by cuproptosis.

Acute tubular necrosis mediates acute kidney injury (AKI) and nephron loss1, the hallmark of end-stage renal disease2–4. For decades, it has been known that female kidneys are less sensitive to AKI5,6. Acute tubular necrosis involves dynamic cell death propagation by ferroptosis along the tubular compartment7,8. Here we demonstrate abrogated ferroptotic cell death propagation in female kidney tubules. 17β-oestradiol establishes an anti-ferroptotic state through non-genomic and genomic mechanisms. These include the potent direct inhibition of ferroptosis by hydroxyoestradiol derivatives, which function as radical trapping antioxidants, are present at high concentrations in kidney tubules and, when exogenously applied, protect male mice from AKI. In cells, the oxidized hydroxyoestradiols are recycled by FSP19,10, but FSP1-deficient female mice were not sensitive to AKI. At the genomic level, female ESR1-deficient kidney tubules partially lose their anti-ferroptotic capacity, similar to ovariectomized mice. While ESR1 promotes the anti-ferroptotic hydropersulfide system, male tubules express pro-ferroptotic proteins of the ether lipid pathway which are suppressed by ESR1 in female tissues until menopause. In summary, we identified non-genomic and genomic mechanisms that collectively explain ferroptosis resistance in female tubules and may function as therapeutic targets for male and postmenopausal female individuals. 17β-oestradiol establishes an anti-ferroptotic state in female kidney tubules.

Ferroptosis, an iron-dependent form of regulated cell death, has been identified as a key factor in the pathogenesis and progression of various cardiovascular diseases (CVDs). The molecular basis of ferroptosis involves lipid peroxidation, iron dysregulation, and antioxidant pathways. Accumulating evidence suggests that ferroptosis is associated with various CVDs, including atherosclerosis, myocardial ischaemia-reperfusion injury, cardiac hypertrophy, and diverse cardiomyopathies. In addition to ferroptosis inhibitors, several novel ferroptosis-targeting therapies have emerged, including new uses for existing drugs, plant-derived compounds, and targeted drug delivery. This review synthesizes current knowledge, identifies research gaps to summarize the complex regulatory mechanisms of ferroptosis, and provides an overview of its functions in CVDs. The innovative therapeutic strategies targeting ferroptosis may lead to a paradigm change in the treatment of CVDs.

Constitutive activation of STING by gain-of-function mutations triggers manifestation of the systemic autoinflammatory disease STING-associated vasculopathy with onset in infancy (SAVI). In order to investigate the role of signaling by tumor necrosis factor (TNF) in SAVI, we used genetic inactivation of TNF receptors 1 and 2 in murine SAVI, which is characterized by T cell lymphopenia, inflammatory lung disease, and neurodegeneration. Genetic inactivation of TNFR1 and TNFR2, however, rescued the loss of thymocytes, reduced interstitial lung disease, and neurodegeneration. Furthermore, genetic inactivation of TNFR1 and TNFR2 blunted transcription of cytokines, chemokines, and adhesions proteins, which result from chronic STING activation in SAVI mice. In addition, increased transendothelial migration of neutrophils was ameliorated. Taken together, our results demonstrate a pivotal role of TNFR signaling in the pathogenesis of SAVI in mice and suggest that available TNFR antagonists could ameliorate SAVI in patients.

This Part 2 of a bipartite review commences with the delineation of a conceptual model outlining the fundamental role of injury-induced regulated cell death (RCD) in the release of DAMPs that drive innate immune responses involved in early inflammation-related allograft dysfunction and alloimmune-mediated allograft rejection. In relation to this topic, the focus is on the divergent role of donor and recipient dendritic cells (DCs), which become immunogenic in the presence of DAMPs to regulate alloimmunity, but in the absence of DAMPs acquire tolerogenic properties to promote allotolerance. With respect to this scenario, proposals are then made for leveraging RCD and DAMPs as biomarkers during normothermic regional perfusion (NRP) and normothermic machine perfusion (NMP) of transplant organs from DCD donors, a strategy poised to significantly enhance current policies for assessing donor organ quality. The focus is then on the ambitious goal to target RCD and DAMPs therapeutically during NRP and NMP, aiming to profoundly suppress subsequently early allograft inflammation and alloimmunity in the recipient. This strategic approach seeks to prevent the activation of intragraft innate immune cells including DCs during donor organ reperfusion in the recipient, which is driven by ischemia/reperfusion injury-induced DAMPs. In this context, available inhibitors of various types of RCD, as well as scavengers and inhibitors of DAMPs are highlighted for their promising therapeutic potential in NRP and NMP settings, building on their proven efficacy in other experimental disease models. If successful, this kind of therapeutic intervention should also be considered for application to organs from DBD donors. Finally, drawing on current global insights into the critical role of RCD and DAMPs in driving innate inflammatory and (allo)immune responses, targeting their inhibition and/or prevention during normothermic perfusion of transplant organs from DCD donors - and potentially DBD donors - holds the transformative potential to not only alleviate transplant dysfunction and suppress allograft rejection but also foster allograft tolerance.

This Part 1 of a bipartite review commences with a succinct exposition of innate alloimmunity in light of the danger/injury hypothesis in Immunology. The model posits that an alloimmune response, along with the presentation of alloantigens, is driven by DAMPs released from various forms of regulated cell death (RCD) induced by any severe injury to the donor or the donor organ, respectively. To provide a strong foundation for this review, which examines RCD and DAMPs as biomarkers and therapeutic targets in normothermic regional perfusion (NRP) and normothermic machine perfusion (NMP) to improve outcomes in organ transplantation, key insights are presented on the nature, classification, and functions of DAMPs, as well as the signaling mechanisms of RCD pathways, including ferroptosis, necroptosis, pyroptosis, and NETosis. Subsequently, a comprehensive discussion is provided on major periods of injuries to the donor or donor organs that are associated with the induction of RCD and DAMPs and precede the onset of the innate alloimmune response in recipients. These periods of injury to donor organs include conditions associated with donation after brain death (DBD) and donation after circulatory death (DCD). Particular emphasis in this discussion is placed on the different origins of RCD-associated DAMPs in DBD and DCD and the different routes they use within the circulatory system to reach potential allografts. The review ends by addressing another particularly critical period of injury to donor organs: their postischemic reperfusion following implantation into the recipient—a decisive factor in determining transplantation outcome. Here, the discussion focuses on mechanisms of ischemia-induced oxidative injury that causes RCD and generates DAMPs, which initiate a robust innate alloimmune response.

Regulated necrosis is a known direct consequence of activation of the necroptosis- and pyroptosis- pathways, but may also result from apoptosis in a process referred to as secondary necrosis. Apoptosis is well understood to be mediated by caspase activation, but the mechanisms that lead to plasma membrane rupture in secondary necrosis remain considerably obscure. Recent data suggested a caspase-mediated cleavage of gasdermin E (GSDME), a member of the gasdermin family. Here, apoptosis induced by diphtheria toxin (DT) is employed as a novel tool to study secondary necrosis. In addition, cisplatin and anti-Fas monoclonal antibody Jo2 are employed to study secondary necrosis in cell culture and in vivo, respectively. Despite prominent, yet epiphenomenal cleavage of GSDME, it is demonstrated that silencing or CRISPR/Cas9-mediated knockout of GSDME does not compromise the kinetics of secondary necrosis induced by DT or cisplatin. During Jo2-induced acute liver toxicity in mice, GSDME expressed in the necrotic liver is detected predominantly in its uncleaved form. In conclusion, the hypothesis of GSDME to be a central mediator of secondary necrosis in these model systems is disproved.

Several forms of regulated necrosis contribute to the pathogenesis of crystal nephropathy, however, the role of pyroptosis, an inflammatory form of cell death involving the formation of gasdermin-D pores in internal and external cell membranes, in this condition remains unknown. Our transcriptional and histological analyses suggest that Gsdmd in tubulointerstitital cells may contribute to the pathogenesis of chronic oxalate nephropathy. However, genetic deletion of Gsdmd exacerbated oxalate nephropathy in mice in association with enhanced CaOx crystal deposition and accelerated tubular epithelial cell injury. Pharmacological inhibition of necroptosis reversed this effect. Indeed, Gsdmd-/- bone marrow-derived macrophages were more prone to undergo necroptosis when stimulated with CaOx crystals compared to their wildtype counterparts. We conclude that gasdermin D suppresses the necroptosis pathway, which determines the outcome of oxalate nephropathy-related nephrocalcinosis.

Necroptosis is a caspase-independent modality of cell death implicated in many inflammatory pathologies. The execution of this pathway requires the formation of a cytosolic platform that comprises RIPK1 and RIPK3 which, in turn, mediates the phosphorylation of the pseudokinase MLKL (S345 in mouse). The activation of this executioner is followed by its oligomerisation and accumulation at the plasma-membrane where it leads to cell death via plasma-membrane destabilisation and consequent permeabilisation. While the biochemical and cellular characterisation of these events have been amply investigated, the study of necroptosis involvement in vivo in animal models is currently limited to the use of Mlkl-/- or Ripk3-/- mice. Yet, even in many of the models in which the involvement of necroptosis in disease aetiology has been genetically demonstrated, the fundamental in vivo characterisation regarding the question as to which tissue(s) and specific cell type(s) therein is/are affected by the pathogenic necroptotic death are missing. Here, we describe and validate an immunohistochemistry and immunofluorescence-based method to reliably detect the phosphorylation of mouse MLKL at serine 345 (pMLKL-S345). We first validate the method using tissues derived from mice in which Caspase-8 (Casp8) or FADD are specifically deleted from keratinocytes, or intestinal epithelial cells, respectively. We next demonstrate the presence of necroptotic activation in the lungs of SARS-CoV-infected mice and in the skin and spleen of mice bearing a Sharpin inactivating mutation. Finally, we exclude necroptosis occurrence in the intestines of mice subjected to TNF-induced septic shock. Importantly, by directly comparing the staining of pMLKL-345 with that of cleaved Caspase-3 staining in some of these models, we identify spatio-temporal and functional differences between necroptosis and apoptosis supporting a role of RIPK3 in inflammation independently of MLKL versus the role of RIPK3 in activation of necroptosis.

Ferroptosis was recently identified as a non-apoptotic, iron-dependent cell death mechanism that is involved in various pathologic conditions. There is first evidence for its significance also in the context of islet isolation and transplantation. Transplantation of pancreatic human islets is a viable treatment strategy for patients with complicated diabetes mellitus type 1 (T1D) that suffer from severe hypoglycemia. A major determinant for functional outcome is the initial islet mass transplanted. Efficient islet isolation procedures and measures to minimize islet loss are therefore of high relevance. To this end, better understanding and subsequent targeted inhibition of cell death during islet isolation and transplantation is an effective approach. In this study, we aimed to elucidate the mechanism of ferroptosis in pancreatic islets. Using a rodent model, isolated islets were characterized relating to the effects of experimental induction (RSL3) and inhibition (Fer1) of ferroptotic pathways. Besides viability, survival, and function, the study focused on characteristic ferroptosis-associated intracellular changes such as MDA level, iron concentration and the expression of ACSL4. The study demonstrates that pharmaceutical induction of ferroptosis by RSL3 causes enhancement of oxidative stress and leads to an increase of intracellular iron, zinc and MDA concentration, as well as the expression of ACSL4 protein. Consequently, a massive reduction of islet function, viability, and survival was found. Fer1 has the potential to inhibit and attenuate these cellular changes and thereby protect the islets from cell death.

Ferroptosis is a regulated cell death modality triggered by iron-dependent lipid peroxidation. Ferroptosis plays a causal role in the pathophysiology of various diseases, making it a promising therapeutic target. Unlike all other cell death modalities dependent on distinct signaling cues, ferroptosis occurs when cellular antioxidative defense mechanisms fail to suppress the oxidative destruction of cellular membranes, eventually leading to cell membrane rupture. Physiologically, only two such surveillance systems are known to efficiently prevent the lipid peroxidation chain reaction by reducing (phospho)lipid hydroperoxides to their corresponding alcohols or by reducing radicals in phospholipid bilayers, thus maintaining the integrity of lipid membranes. Mechanistically, these two systems are linked to the reducing capacity of glutathione peroxidase 4 (GPX4) by consuming glutathione (GSH) on one hand and ferroptosis suppressor protein 1 (FSP1, formerly AIFM2) on the other. Notably, the importance of ferroptosis suppression in physiological contexts has been linked to a particular vulnerability of renal tissue. In fact, early work has shown that mice genetically lacking Gpx4 rapidly succumb to acute renal failure with pathohistological features of acute tubular necrosis. Promising research attempting to implicate ferroptosis in various renal disease entities, particularly those with proximal tubular involvement, has generated a wealth of knowledge with widespread potential for clinical translation. Here, we provide a brief overview of the involvement of ferroptosis in nephrology. Our goal is to introduce this expanding field for clinically versed nephrologists in the hope of spurring future efforts to prevent ferroptosis in the pathophysiological processes of the kidney.

Thrombotic microangiopathy (TMA) is characterized by immunothrombosis and life-threatening organ failure, but the precise underlying mechanism driving its pathogenesis remains elusive. In this study, we hypothesized that gasdermin D (GSDMD), a pore-forming protein serving as the final downstream effector of pyroptosis/interleukin (IL)-1β pathway, contributes to TMA and its consequences by amplifying neutrophil maturation and subsequent necrosis. Using a murine model of focal crystalline TMA, we found that Gsdmd-deficiency ameliorated immunothrombosis, acute tissue injury and failure. Gsdmd-/- mice exhibited a decrease in mature IL-1β, as well as in neutrophil maturation, β2 integrin activation, and recruitment to TMA lesions, where they formed reduced neutrophil extracellular traps both in arteries and interstitial tissue. The GSDMD inhibitor disulfiram dose-dependently suppressed human neutrophil pyroptosis in response to cholesterol crystals. Experiments with GSDMD-deficient human induced pluripotent stem cell-derived neutrophils confirmed the involvement of GSDMD in neutrophil β2 integrin activation, maturation as well as pyroptosis. Both prophylactic and therapeutic administration of disulfiram protected mice from focal TMA, acute tissue injury and failure. Our data identify GSDMD as a key mediator of focal crystalline TMA and its consequences: ischemic tissue infarction and organ failure. GSDMD could potentially serve as a therapeutic target for systemic forms of TMA.

Macroautophagy/autophagy is a complex degradation process with a dual role in cell death that is influenced by the cell types that are involved and the stressors they are exposed to. Ferroptosis is an iron-dependent oxidative form of cell death characterized by unrestricted lipid peroxidation in the context of heterogeneous and plastic mechanisms. Recent studies have shed light on the involvement of specific types of autophagy (e.g. ferritinophagy, lipophagy, and clockophagy) in initiating or executing ferroptotic cell death through the selective degradation of anti-injury proteins or organelles. Conversely, other forms of selective autophagy (e.g. reticulophagy and lysophagy) enhance the cellular defense against ferroptotic damage. Dysregulated autophagy-dependent ferroptosis has implications for a diverse range of pathological conditions. This review aims to present an updated definition of autophagy-dependent ferroptosis, discuss influential substrates and receptors, outline experimental methods, and propose guidelines for interpreting the results.Abbreviation: 3-MA:3-methyladenine; 4HNE: 4-hydroxynonenal; ACD: accidentalcell death; ADF: autophagy-dependentferroptosis; ARE: antioxidant response element; BH2:dihydrobiopterin; BH4: tetrahydrobiopterin; BMDMs: bonemarrow-derived macrophages; CMA: chaperone-mediated autophagy; CQ:chloroquine; DAMPs: danger/damage-associated molecular patterns; EMT,epithelial-mesenchymal transition; EPR: electronparamagnetic resonance; ER, endoplasmic reticulum; FRET: Försterresonance energy transfer; GFP: green fluorescent protein;GSH: glutathione;IF: immunofluorescence; IHC: immunohistochemistry; IOP, intraocularpressure; IRI: ischemia-reperfusion injury; LAA: linoleamide alkyne;MDA: malondialdehyde; PGSK: Phen Green™ SK;RCD: regulatedcell death; PUFAs: polyunsaturated fatty acids; RFP: red fluorescentprotein;ROS: reactive oxygen species; TBA: thiobarbituricacid; TBARS: thiobarbituric acid reactive substances; TEM:transmission electron microscopy.